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Nonspherical Nanocapsules as Long-Circulating Drug Delivery

Systems
Yunxin Xiao, Angel Tan, Alexander W. Jackson, and Ben J. Boyd*

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ABSTRACT: Polymeric nanocapsules have been studied as drug

delivery carriers for a great variety of drugs for more than 30 years.
In particular, nonspherical polymeric nanoparticles with solid cores
are highlighted for their capability to influence cellular uptake and
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circulation time in the bloodstream. However, the promising

combination of nonspherical morphology and the existence of an
internal cavity to encapsulate a high dose of pharmaceutical actives
has not yet been fully achieved. This review systematically
addresses the development of nonspherical nanocapsules with a
cross-linked shell or self-assembled membrane (polymersomes).
Ellipsoidal, tetrahedral, cubic, protrusion-shaped, dumbbell-
shaped, and snowman-shaped nanocapsules are discussed. The
relationship between the formed morphologies and the use of different templates, monomers, or methods of polymerization are
elucidated for future design of more functional shapes.

1. INTRODUCTION approach can be effective for drugs with low stability in

Synthetic polymers are incredibly versatile materials and have
been widely adopted as building blocks to form drug delivery
vehicles, including polymeric micelles,1 dendrimers,2 bioconju-
gates,3 biodegradable platforms,4 or pH-responsive carriers.5
This widespread interest is attributable to established methods
of synthesis, ease of surface functionalization, versatility in
structure, and cost-effectiveness of raw materials. Polymeric
nanocapsules are defined as core−shell nanoparticles compris-
ing an internal cavity surrounded by a polymeric membrane.6,7
Polymeric nanocapsules can be formed either by the self-
assembly (physical assembly) of amphiphilic multiblock
copolymers affording polymersomes or via templation
methods, which afford a covalently cross-linked polymeric
shell, these two approaches are outlined in Figure 1. Three
types of polymeric materials are outlined in Figure 1: solid
polymeric nanoparticles/unimolecular micelles, self-assembled
polymersomes, and cross-linked nanocapsules. A solid polymer
nanoparticle can encapsulate drug molecules in the internal
matrix of the particle, or in the case of unimolecular micellar
structures, a preference for the hydrophobic core is often seen
for poorly soluble drugs. This is the simplest form of polymeric
drug delivery vehicle out of the three and requires the least
engineering. The drawback is the lack of an internal aqueous
cavity making it difficult to encapsulate hydrophilic drugs and a
limited capacity for drug encapsulation making these systems
inappropriate where high drug doses are required. Drug
loading is alternatively achieved by synthetically attaching the
drug molecule to the monomer units by covalent bonds. This
aqueous solution, and when the polymer chain folds in to form
the solid particle, a portion of the drug molecules would be
contained inside the particle and only released when the
particle is degraded. Because of the lack of internal cavity, solid
polymeric nanoparticles are not in the scope of this review.
For the other two types of polymeric drug delivery systems
in Figure 1, polymersomes and cross-linked nanocapsules, a
large variety of substances, including pharmaceutical actives in
liquid or solid form, can be encapsulated within the cavity.8
Methods of encapsulation include synthetic attachment of
drugs to the polymeric chain similar to the aforementioned
example of solid polymeric nanoparticles, and passive or active
loading into the internal cavity of polymersomes or the vesicle
template for cross-linked nanocapsules. Passive loading of drug
into the core of a membrane structure occurs simply by
inclusion in the aqueous fluid encapsulated upon formation of
the particle, analogous to passive loading of drugs into
liposomes. Active loading occurs after particle formation,
driven by controlling conditions such that the solubility of the
drug in the internal compartment is greater than outside the
Received: October 18, 2021
Revised: January 15, 2022
Published: March 1, 2022

© 2022 American Chemical Society https://doi.org/10.1021/acs.chemmater.1c03573

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Figure 1. Schematic of three general types of polymeric architectures including solid polymeric nanoparticles, cross-linked nanocapsules usually
made by forming a layer of polymer on the surface of a template, and self-assembled nanocapsules, called polymersomes, made via physically driven
assembly of block copolymers.

Table 1. General Templation Methods Used to Prepare Nanocapsules, Where Sacrificial Templates Required Removal
Postpreparation but the Removal of Hollow Nonsacrificial Templates Is Not Required
Mechanism
Method Procedure of formation Template Refs
nanoprecipitation dissolution of polymer in the organic phase physical oil droplet 12
addition of the organic phase to the aqueous phase (sacrificial)
supersaturation of the polymer in the mixture leads to the formation of nanocapsules
emulsification dissolution of polymer in the organic phase physical oil droplet 13
organic phase is emulsified under vigorous agitation in the aqueous phase (sacrificial)
further addition of water leads to the formation of nanocapsules
polymer coating using a core such as solid nanoparticles, pigments, or nanoemulsions physical solid template 14
deposition of a layer of polymer onto the surface of the core (sacrificial)
layer-by-layer using a colloidal template with a charge on the surface (for example, positive charge) physical solid template 15, 18
deposition of the first layer driven by electrostatic interaction (negatively charged polymer on (sacrificial)
positively charge template)
deposition of the second layer (positive charge)
deposition of several layers of alternating positive and negative charge until obtaining a
satisfactory thickness
sacrificial templates templates include gold or silica rods chemical solid template 19−21
polymer is synthesized, often by free radical or RAFT polymerization, on the surface of the (sacrificial)
solid template, which is subsequently removed after formation of the polymer
nonsacrificial small molecule surfactant liposomes or vesicles chemical hollow template 16,
templates polymer is synthesized, often by free radical or RAFT polymerization, on the surface of the (nonsacrificial) 22−26
hollow template which does not have to be removed after formation of the polymer

membrane, often enabled by ion-pairing or a gradient in pH.
For cross-linked nanocapsules, postloading of cargo after
formation is difficult because the diffusion through the cross-
linked shell is intentionally extremely slow. Passive loading is,
therefore, usually the method of choice but potential
degradation of the cargo during the polymerization process
needs to be considered and minimized.
In terms of drug release from the carriers, studies have
shown that encapsulated drugs were released at a slower rate
from polymeric nanocapsules9 compared to other types of
small molecule surfactant vesicles because of the inertness of
the cross-linked polymeric layer to enzymatic activity,
sensitivity to pH, and premature release of drug upon dilution
by body fluid. The type and thickness of the polymeric shell
dictated different rates of release of drug,10 which can help to
avoid side effects or tissue irritation by decreasing nonspecific
release of drug before reaching the target site. To achieve a
controlled release of drug from polymersomes or cross-linked
nanocapsules, stimuli-responsive functional groups can be
incorporated into the polymer chain. Certain linkers, such as
esters, can also be incorporated into the cross-linked shell and
so the release of drug is controlled by the rate of hydrolysis of
the ester bond after administration.
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Premature disassembly of polymersomes might occur before
the particles reach the diseased site, which was similar to
physically assembled small molecule vesicles. As such polymer
scientists have looked to prepare polymeric nanocapsules with
covalently cross-linked polymeric shells. A variety of
templation methods are used to prepare polymeric nano-
capsules depending on the properties of the polymeric material
utilized and the intended cargo.11 The most common form of
template is an oil droplet suspended in an aqueous media. The
diverse templation methodologies can be classified as either
physical formation (i.e., preformed polymers assembly at the
surface of the template) or chemical formation (polymer
chains are prepared during the nanocapsule formation).
Further classification can characterize these methodologies as
either sacrificial templation (template does not possess a void
and must be removed after polymer shell formation) or
nonsacrificial templation (template possess a void and does not
need to be removed after polymer shell formation). These
methods include nanoprecipitation,12 emulsification,13 poly-
mer coating,14 layer-by-layer,15 and vesicle-templated meth-
ods,16 as summarized in Table 1.
In the nanoprecipitation method, nanocapsules are formed
when an organic phase is added to an aqueous phase, where
supersaturation of the polymer in the solvent mixture leads to
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Table 2. Nanocapsules Used as Drug Delivery Systems with Varying Functionalities

Function Example drugs Method and improvement References
encapsulate poorly xanthone solvent displacement method: optimize the solubility of hydrophobic drugs in the oily core by selecting 33, 34, 40
water-soluble drugs griseofulvin the best oil and water composition
triamcinolone the maximum concentration of xanthone reached 600 μg/mL in the nanocapsules which was
acetonide therapeutically effective
sustained-release drug photosensitizer, cross-linked polymeric shells are particularly effective in retaining active drug substances compared to 9, 41−44
carriers IR-780 self-assembled polymersomes or nanospheres because of their more compact structure
atovaquone the thickness of a lipid bilayer is approximately 3 nm while a cross-linked polymeric shell, depending on
the level of cross-linking, could reach 30−40 nm
nanocapsules made of polycaprolactone slowed down the release of 80% of the encapsulated
photosensitizer, IR-780, for 10 h compared to nanospheres
atovaquone-loaded nanocapsules made of poly(lactic acid) were able to retain approximately 75% of the
active compound within 4 months
tunable drug delivery 4-nitroanisole the thickness of the cross-linked shell could be modified to control the release rate of drug substances by 10, 11
systems using different polymeric materials, polymerization reactions or preparation methods
the drug needed to penetrate across a thicker polymer membrane leading to a slower release profile when
higher composition of polymer was used
stimuli-responsive doxorubicin responding to triggers including pH, magnetic field, light or temperature 45, 46
drug delivery under relevant triggers, polymeric shells undergo structural changes including degradation, pore
systems formation, and phase transitions to release the cargo at targeted sites
doxorubicin was loaded into poly(styrenesulfonate) (PSS)-doped calcium carbonate (CaCO3) particles
coated with metal-polyphenol coordination films at pH 8
almost 62% and 53% of DOX were released from the capsules within the first 48 h at pH 5 and pH 6
while less than 30% was released at pH 7.4

its precipitation at the solvent−water interface producing
nanocapsules. In the emulsification method, the organic phase
containing the polymer is emulsified under vigorous agitation
in the aqueous phase and the subsequent addition of water
causes the formation of nanocapsules, again at the solvent−
water interface. These two methods are used to encapsulate a
wide range of cargos but the harsh conditions needed to agitate
the formulation and remove all residual solvents limit their
biological applications.17 Polymer coating involves deposition
of a layer of polymer onto the surface of nanoparticles
including solid nanoparticles, pigments, or nanoemulsions,
these methods often require the removal of the template post-
nanocapsule formation. Layer-by-layer and other templation
methods are similar to polymer coating in the sense of the
deposition of preformed polymers but differ in the types of
templates and the driving force used. In the layer-by-layer
method, the polymer layers are adsorbed on the colloidal
template driven by electrostatic attraction and several layers
could be deposited with alternating positive and negative
charges to obtain a thick polymeric shell.18 In other templation
methods, sacrificial templates, such as gold19,20 or silica rods,21
or nonsacrificial biologically compatible templates, such as
liposomes22 or surfactant vesicles,23,24 are used. Typically,
polymers are synthesized (chemical formation) at the interface
of the template and the dispersant using a wide range of
polymerization reactions including free radical polymer-
ization25 and reversible addition−fragmentation chain transfer
(RAFT) polymerization.26 Harsh processing conditions are
avoided by using water as the dispersant throughout the
formation of nanocapsules which enables wider biological
applications. Using a nonsacrificial hollow template to form
nanocapsules allows pre-encapsulation of cargo to maximize
the encapsulation efficiency. Nonsacrificial templates also
negate the costly and laborious requirement of template
removal while the final morphology of nanocapsules are being
controlled.
Most research into nanocapsules has focused on modifica-
tion of the composition of polymers and advances in synthetic
routes and surface functionality. However, while the shape of
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solid nanoparticles can play a significant role in the efficiency
of drug delivery, cellular interactions, and presystemic
excretion,27,28 the shape of polymeric nanocapsules has not
been extensively explored. There were a few reports showing
nanocapsules having different interactive behaviors with
different cells, resulting in changes in systematic circulation
time, amount of accumulation at the disease site, cellular
uptake and consequently therapeutic effects.29,30 In this review,
the preparation of nonspherical polymeric nanocapsules using
different methods is systematically presented. Despite the great
variety of nonspherical solid nanoparticles in the literature,
they are excluded from the definition of nanocapsules due to
the lack of internal cavity for encapsulation, hence they are out
of the scope of this review. In the biological context, research
into the cellular interactions of nonspherical nanocapsules are
still in its infancy. Therefore, influence of morphology on
interactions including circulation time and therapeutic
efficiency of nonspherical particles are gained from the
investigations using nonspherical polymeric nanoparticles in
general.
2. NANOCAPSULES AS DRUG DELIVERY SYSTEMS
Spherical nanocapsules have been widely exploited as drug
delivery systems because of their versatility in encapsulating
poorly water-soluble (oily core) and water-soluble (aqueous
core) drugs, protection of active drugs against degradation
factors, such as light, pH, or temperature, and their capacity to
act as sustained, controlled, and stimuli-responsive release
carriers (Table 2). As compared to other widely used particle-
based carriers, such as micelles or nanospheres, nanocapsules
are more resistant to structural changes in physiological
conditions.31 Self-assembled structures, such as liposomes, are
more prone to disassembly upon administration, while the
cross-linked membrane of nanocapsules can be designed to
only be digested by certain groups of enzymes (for example,
hydrolysis reaction for the ester linker) leading to a slow
disassembly. A thick membrane also provides better protection
for the cargo. Consequently, range of synthetic polymers,
including poly(acrylic acid),32 poly(lactic acid),33 poly(lactic-
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Figure 2. Examples of using nanocapsules as drug delivery systems for various purposes: (a) To encapsulate poorly water-soluble drug such as
xanthone (XAN) to reach a therapeutically effective concentration. Adapted with permission from ref 40. Copyright 2005 Elsevier. (b and c)
Tunable drug delivery system to decrease the rate of drug release by increasing the weight of poly(lactic acid) (PLA) from 1.5 to 2.5 g. Legends
represent different structures: I, with a liquid core containing the active ingredient; II, active ingredient is contained in the central of the continuous
polymeric matrix; III, active ingredient is evenly distributed in the polymeric matrix. Adapted with permission from ref 10. Copyright 2002 Elsevier.
(d) Act as a pH-triggered stimuli-responsive doxorubicin delivery system, where higher percentage of doxorubicin was released at lower pH which
is representative of the intracellular environment of cancer cells, instead of the high pH of the extracellular fluid. Adapted with permission from ref
45. Copyright 2015 John Wiley and Sons.

co-glycolic acid),9 poly-ε-caprolactone,34,35 and poly(ethylene
glycol),36 have been used to deliver a wide range of
pharmaceutical actives including ciprofloxacin,37 tamoxifen,38
penicillin G,39 and DNA.36
In applications for poorly water-soluble drugs, nanocapsules
have been shown to improve the bioavailability of
pharmaceutical actives, such as xanthone,40 griseofulvin,34
and triamcinolone acetonide,33 to improve their bioavailability.
The nanocapsules were prepared using the solvent displace-
ment method and the solubility of hydrophobic drugs in the
oily core was optimized through selection of the best oil and
water composition.41 Taking xanthones as an example, they are
a group of heterocyclic compounds with dibenzo-γ-pyrone
backbone, which present anti-inflammatory and immunomo-
dulatory activities. However, their poor aqueous solubility
limited their biological applications. In the study by Teixeira et
al., xanthone was first dissolved in Myritol 318 oil which was
then added to the acetone solution of poly(DL-lactide-co-
glycolide) (PLGA) and soybean lecithin. The solution was
poured into aqueous solution of Pluronic F-68 under moderate
stirring to form nanocapsules. The maximum concentration of
xanthone reached 600 μg/mL in the nanocapsules, which was
therapeutically effective (Figure 2a).40
Nanocapsules are also used as sustained-release drug carriers
for prolonged release applications. In general, cross-linked
polymeric shells are particularly effective in retaining active
drug substances compared to self-assembled polymersomes or
nanospheres because of their more compact structure.41 It has
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been observed that most active drug substances showed an
initial release phase that could be attributed to the high
concentration gradient across the shell or release of drug
located near the surface of nanocapsules,33 followed by a
second phase corresponding to the slow diffusion of drug from
the inner core or degradation of the polymer over time.36,47
Individual drug release profiles from different polymeric
nanocapsules depend on a variety of factors, such as the
intrinsic physicochemical properties, concentration of the
drugs, the degradability, types, molecular weight of the
polymeric materials, the thickness, level of cross-linking, and
size distribution of the final nanocapsules.11 Particularly, there
has been a clear trend of reduced rate of drug release from
nanocapsules with thicker polymeric shells. The thickness of a
lipid bilayer membrane is around 3 nm,42 while the thickness
of a polymeric shell, which is highly dependent on the types of
material and level of cross-linking, of similar particle size
distribution, could be around 30−40 nm.43 In the study by
Bazylińska et al., nanospheres and nanocapsules made of
polycaprolactone showed that nanocapsules slowed the release
of 80% of the encapsulated photosensitizer, IR-780, for 10 h
compared to nanospheres. This was due to the enclosure of
hydrophobic IR-780 in the oily core of the nanocapsules
compared to only in the polymeric matrix of nanospheres.44
Cauchetier et al. also showed that atovaquone-loaded nano-
capsules made of poly(lactic acid) were able to retain
approximately 75% of the active compound within 4 months.9
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Chemistry of Materials
Nanocapsules are also used as tunable drug delivery systems
to control the release rate of drugs. The thickness of the cross-
linked shell can be modified to control the release rate of drug
substances by using different polymeric materials, polymer-
ization reactions or preparation methods.11 When 1 g of extra
poly(lactic acid) material was used, 15% less 4-nitroanisole was
released from nanocapsules in the same duration (Figure 2b
and c). The drug needed to penetrate across a thicker polymer
membrane leading to a slower release profile when higher
composition of polymer was used.10
Polymeric nanocapsules are also used as stimuli-responsive
drug delivery systems to respond to triggers including pH,
magnetic field, light and temperature. With the presence of
certain triggers, polymeric shells undergo structural changes
including degradation, pore formation, and phase transitions to
release the cargo at targeted sites. Ping et al. made use of the
different pH environments between extracellular fluids (pH
7.4), tumor tissues (pH 5.7−6.8), and the acidic environment
inside endosomal (pH 5.5−6.0) and lysosomal (pH 4.5−5.0)
compartments to control the ionization state of polymers to
target doxorubicin (DOX) to HeLa and MB231 cells.
Doxorubicin was loaded into poly(styrenesulfonate) (PSS)-
doped calcium carbonate (CaCO3) particles to form PSS-DOX
complexes with electrostatic interaction, hydrogen bonding
and hydrophobic interaction. The DOX-coated CaCO 3
templates were then coated with metal-polyphenol coordina-
tion films at pH 8. Almost 62% and 53% of DOX were released
from the capsules within the first 48 h at pH 5 and pH 6 while
less than 30% was released at pH 7.4 (Figure 2d). The release
was attributed to the reduced hydrogen bonding between
DOX and PSS leading to easier diffusion of DOX through the
permeable shells at low pH.45 Recently, polymersome
nanoreactors were developed to be switched on by visible
light and self-revert back to their inactive state in the darkness.
Donor−acceptor Stenhouse adducts (DASAs), photochromic
compounds able to convert to cyclic zwitterionic form from
open form under light of a certain wavelength, was used to
increase the polarity of the polymersome membranes from a
nonpolar triene-enol form to a cyclopentenone. Release of
payload started in the presence of light and stopped when the
light was turned off, opening up opportunities for on-demand
drug delivery.46
Functionalized nanocapsules used as stimuli-responsive drug
delivery systems, either with covalently cross-linked shell or
self-assembled membranes, have been reviewed in-
depth.18,48,49 Most studies focused on increasing encapsulation
efficiency of cargo, improving the surface functionality and
advancing synthetic routes to achieve targeted delivery of
nanocapsules. However, the role of geometry of the nano-
capsules was not addressed. Traditional preparation meth-
ods,11 such as solvent displacement and emulsion evaporation
approaches, have limited the production of nonspherical
nanocapsules.
3. NONSPHERICAL POLYMERIC NANOCAPSULES
The shape of nanoparticles has been suggested to play an
important role in effective drug delivery by influencing the
cellular uptake by macrophages, biodistribution, eventually
cytotoxicity and therapeutic effects.28,50,51 In the study by
Champion et al., it was found that macrophages internalized
spherical particles at a faster rate than elliptical polystyrene
particles. For elliptical particles, the efficiency of uptake was
also determined by the point of contact. If the particles came
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into contact with the macrophage at the major axis, the uptake
was much slower compared to when they came into contact at
the minor axis (Figure 3).51 The study demonstrated the
Figure 3. Example of the shape of nanoparticles influencing the
cellular internalization of macrophages observed via scanning electron
microscopy. Micrographs a−c of cells and particles were colored
brown and purple, respectively. (a) The cell body can be seen at the
end of an opsonized elliptical disk, and the membrane has progressed
down the length of the particle. (Scale bar: 10 μm.) (b) A cell has
attached to the flat side of an opsonized elliptical disk and has spread
on the particle. (Scale bar: 5 μm.) (c) An opsonized spherical particle
has attached to the top of a cell, and the membrane has progressed
over approximately half the particle. (Scale bar: 5 μm.) Adapted with
permission from ref 51. Copyright 2006 National Academy of
Sciences, U.S.A.
potential to modify cellular uptake by engineering the
morphology of polymeric particles. The two types of
nanocapsules addressed in this review, cross-linked nano-
capsules and self-assembled polymersomes, are formed via
different methods. Functionalized block copolymers are usually
preformed before the self-assembly step to form polymer-
somes. Therefore, the morphology of polymersomes is
dependent on the conditions introduced in the self-assembly
process (such as solvent-switch method52 and osmotic pressure
method53). For cross-linked nanocapsules, polymerization
reaction with the formation of covalent bonds is the
mechanism of formation and so the types and conditions of
polymerization, the interplay between the template and the
polymerized material are all significant in the determination of
resulting morphology. In the context of drug delivery, both
types of nanocapsules are of importance for researchers
because they can provide different rates of drug release, with
self-assembled polymersomes that possess a noncovalently
bound, leakier membrane usually having a faster release rates
compared to covalently cross-linked nanocapsules.
In the past, preparation of nonspherical nanocapsules usually
rely on the use of a nonspherical solid inorganic template, such
as silica,54 iron oxide,55 or gold nanoparticles.55 However, the
core-removal step, which usually involves the use of organic
solvents or harsh processing methods, could compromise or
completely damage the activity of the cargo or the polymer
itself. Therefore, two alternative approaches were developed
for nanocapsule formation; for nonspherical polymersomes,
block copolymers were preconstructed prior to the self-
assembly step and no template-removal was needed and so the
activity of the pharmaceutical cargo was protected. A range of
functional groups could be synthesized in the copolymer, and
so, a wide range of functionalities could be incorporated.
Second, the development of the vesicle-templated method
provided opportunities to use soft and biologically compatible
nonspherical vesicles as templates to form nonspherical
nanocapsules to prevent the need for template removal and
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Table 3. Summary of Nonspherical Nanocapsules of Different Morphologies via Various Polymerization Techniquesa

a
Abbreviations: AA, acrylic acid; BA, butyl acrylate; CdCO3, cadmium carbonate; DVB, divinylbenzene; HCl, hydrochloric acid; HF, hydrofluoric
acid; MMA, methyl methacrylate; MPS, 3-(trimethoxysilyl)propyl methacrylate; NIPAM, N-isopropylacrylamide; PDMI, perylene diester
monoimide; PS-co-PMPS, poly(styrene-co-3-(trimethoxysilyl)propyl methacrylate); PS, polystyrene; RAFT, reversible addition−fragmentation
chain-transfer; SiO2, silicon dioxide; SnS, tin sulfide; St, styrene; THF, tetrahydrofuran.
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 Table 4. Advantages and Disadvantages of Different Methods to Prepare Nonspherical Nanocapsules
Methods Morphologies prepared Advantages Disadvantages Refs
hard inorganic ellipsoidal a great variety of shapes the removal of the template requires addition of highly corrosive agents 26, 54, 56
templates relatively stable throughout the polymerization reaction pharmaceutical actives might be deactivated
polymeric material is able to adopt the shape of the template upon coating the template does not have an internal cavity to preload cargo
soft vesicle parachute-shaped material itself is biocompatible so no template removal is needed harder to maintain the nonspherical shape due to their low rigidity 57−60
templates protrusion-shaped vesicular feature allows potential encapsulation of drugs variety of shape is limited because the final shape depends on the kinetics of
polymerization and properties of the formed polymer
Chemistry of Materials

better chance to maintain the bioactivity of the encapsulated cargo

core−shell dumbbell-shaped able to form dual- or multicompartment nanocapsules complex preparation procedure due to the multiple layers 64−67
templates snowman-shaped able to form Janus nanocapsules with materials of distinct properties on each optimization is time-consuming because both properties of the core and the shell
lobe layer need to be precisely designed
a great variety of shapes are available when different materials protrude at
different rates and form various sizes
the size ratio of various lobes can be adjusted by designing the core or shell
material and the kinetics of polymerization
RAFT oligomer ellipsoidal able to drive the polymerization on the surface of the template involves the design of at least two polymerization reactions−the formation of the 26, 59, 60
adsorption RAFT oligomer and the final chain extension/cross-linking reaction
parachute-shaped increase encapsulation of a variety of cargo such as dye by located adsorption need the presence of electrostatic interactions between the vesicle and the
protrusion-shaped separate the process of forming polymeric shell and encapsulating drugs (by the oligomer or attractive forces, otherwise efficiency of adsorption might be low
template) to provide better encapsulation and drug stability
free radical ellipsoidal relatively simple design of reaction lack of control over the molecular weight and distribution of chemical 54, 57, 58,
polymerization parachute-shaped great availability of raw materials composition of the formed polymers 60, 64−67
dumbbell-shaped instant growth of large polymers creating the second partially fused solid inability to form a homogeneous layer of shell

2509
snowman-shaped
RAFT protrusion-shaped able to form a homogeneous polymeric shell by controlling the kinetics of the establishment of the RAFT control requires in-depth understanding of the 26, 59, 60,
polymerization polymerization mechanism 68, 70, 71
homogeneous spherical able to form polymers or oligomers with the designed arrangement and chain type of materials are limited because the selection of monomer needs to be
hollow nanocapsules length matched with the selection of RAFT agent and initiator
layer-by-layer cubic deposited layers adopt the shape of the template need to use electrostatic interactions for each layer and so the selection of 15, 56
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material is relatively limited

tetrahedral does not require the use of harsh procedure such as high temperature which aggregation might occur instead of deposition if the molar ratio is not adjusted
protects the activity of the cargo and optimized
able to deposit multiple layers the thickness of each layer is low and so if a thick shell is required, deposition of
electrostatic interactions are able to hold the multiple layers together with many layers is tedious
relatively high stability
self-assembly ellipsoidal no polymerization is needed further after obtaining the block copolymer careful design of functional groups and hydrophobicity of the polymer is needed 68−72
to successfully prepare nonspherical shapes
faceted large interior volume to encapsulate cargo the prepared nonspherical shape is less stable compared to a cross-linked and
covalently cross-linked shape
tubular self-assembly drives the formation of the structure providing better chance to sensitive to a change of conditions such as solvent, ion strength and introduction
stomatocyte-shaped maintain the bioactivity of drugs compared to polymerization reactions of other chemicals
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Figure 4. (a) Schematic of the preparation of hematite/silica/polymer trilayer hybrids and the corresponding hollow polymer ellipsoidal seeds; (b)
TEM micrograph of hematite/silica/PDVB trilayer hybrids obtained with DVB feed for the polymerization; (c) TEM micrograph of hematite/
silica/PMBAAm trilayer hybrids; (d) TEM micrograph of PDVB hollow ellipsoids; (e) TEM micrograph of hollow PMBAAm ellipsoids with
movable hematite cores. Adapted with permission from ref 54. Copyright 2008 American Chemical Society.

maintain the activity of the encapsulated cargo. Vesicles made
of natural phospholipids or surfactant molecules can also
encapsulate pharmaceutical actives to achieve preloading of
cargo into the nanocapsules. The drawbacks of using these soft
templates are their limited capability to maintain the
nonspherical shape upon changes of conditions, such as pH,
concentration of electrolytes, addition of amphiphilic additives,
or temperature, during the formation of the polymeric shell.
Therefore, in this section, nonspherical nanocapsules with
covalently cross-linked shell formed with inorganic hard
templates (section 3.1), vesicular soft templates (section
3.2), multicompartmental nanocapsules including dumbbell-
shaped, snowman-shaped, Janus nanocapsules (section 3.3)
with dual properties, and self-assembled polymersomes
(section 3.4) are discussed. A summary of various
morphologies are given in Table 3, and the advantages and
disadvantages of different methods of preparation are given in
Table 4, which are each elaborated on in the subsequent
sections.
3.1. Ellipsoidal, Platelet-Shaped, Cubic and Tetrahe-
dral Nanocapsules Prepared with Inorganic Templates.
In this section, using inorganic materials as the template to
construct nonspherical morphologies is discussed, together
with three main shell-forming approaches, namely distillation
precipitation polymerization, controlled RAFT polymerization
and layer-by-layer method. The three methods provided
different driving forces for the polymeric layer to deposit
onto the surface of the templates. In the first example, the
driving force was the capture of monomer radical by a vinyl
group on the surface of the core. In the second and third
examples, electrostatic attraction has been used, either between
the RAFT oligomer and the template, or the template and the
consecutively deposited layers with opposite charges. The
maintenance of the nonspherical shape after template removal,
limitations on the selection of polymers and approaches to
control the thickness are addressed below.
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Ellipsoidal nanocapsules made of divinylbenzene (DVB) or
N,N′-methylenebis(acrylamide) (MBAAm) were prepared via
distillation precipitation polymerization (Figure 4a). The seeds
were unmodified hematite/silica particles and analogues
modified by 3-(methacryloxy)propyl trimethoxysilane (MPS)
(hematite/MPS-modified SiO2), poly(ethylene glycol dime-
thacrylate), and poly(divinylbenzene-co-methacrylic acid).
Hematite (α-Fe2O3) is the most stable form of iron oxide
under ambient conditions with semiconducting properties,
which has been used to control particle shape, size and
magnetic properties. Polymerization was performed in neat
acetonitrile with 2,2′-azobisisobtyronitrile (AIBN) as the
initiator to coat the seeds to form hematite/silica polymer
trilayer hybrids with PDVB (Figure 4b) or PMBAAm (Figure
4c). The driving force for polymer formation around the
template was the capture of DVB oligomer radicals with the aid
of a vinyl group on the surface of the hematite/MPS-modified
silica core−shell particles, which limits the selection of both
monomer and template. However, other hematite/silica/
polymer trilayer hybrid particles with different polarity and
functionality such as the ones with poly(ethylene glycol
dimethacrylate) and poly(divinylbenzene-co-methacrylic acid)
could also be prepared using this method.54
Hollow PDVB ellipsoids were obtained by removal of the
silica midlayer and hematite core with concentrated hydro-
fluoric acid solution (Figure 4d). If dilute hydrofluoric acid was
used instead, only the silica midlayer were removed and hollow
PDVB and PMBAAm ellipsoids with movable hematite cores
were obtained (Figure 4e). The ellipsoidal shape was
maintained after the removal of the template because of the
cross-linking of the polymeric shell. The thickness of the
PDVB shell layer was controlled by altering the type or amount
of monomer feed during the polymerization, including the use
of a cross-linker, ethylene glycol dimethacrylate (EGDMA), a
combination of DVB and MAA or MBAAm.54 The need to use
acetonitrile as the solvent for polymerization and to remove
the hard templates with an acid limited its application in
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Figure 5. (a) Schematic representation of the synthesis of polymer/gibbsite nanocomposite latex particles by aqueous starved feed emulsion
polymerization with the use of RAFT copolymers as stabilizers. (b) Cryo-TEM micrograph of the gibbsite platelets. (c) Cryo-TEM micrographs of
the encapsulated gibbsite particles obtained by RAFT copolymer BA7.5-co-AA10. (d) Single polymer-encapsulated gibbsite platelet obtained by
using RAFT copolymer BA5-co-AA5 and a feed composition ratio of MMA:BA = 10:1 for encapsulation at a higher magnification (black dots in the
frame are 10 nm gold particles added for calibration). Adapted with permission from ref 26. Copyright 2009 American Chemical Society.

Figure 6. SEM images of (a) CdCO3 particles (inset scale bar is 2 μm) and (b) tetrahedral SnS crystals. (c) Confocal microscopy images of cubic
hollow capsules at pH 5 and (d) tetrahedral microcapsules (scale bar 1 μm). Adapted with permission from ref 56. Copyright 2010 John Wiley and
Sons.

encapsulating biological actives. However, the study presented
a method to use the cost-effective iron oxide as an inorganic
template to prepared well-defined ellipsoidal nanocapsules
with controllable size and thickness of the shell. It also
demonstrated the possibility to use different concentrations of
hydrofluoric acid to selectively remove either only the silica
layer of the shell or both the silica and the iron oxide layer for
different functionalities.
In another study, a different polymerization technique,
RAFT polymerization, was used, which provides a different
driving force to attach the polymeric layer onto the surface of
the template. Dibenzyltrithiocarbonate (DBTTC) was used as
the RAFT agent and gibbsite platelets were the template
(Figure 5a). In this example, gibbsite platelets (γ-Al(OH)3)
were used as a model substrate to evaluate the efficiency of the
method to encapsulate clays (Figure 5b) instead of forming
hollow nanocapsules. Therefore, the gibbsite platelet was not
removed at the end. Emulsion polymerization has been used to
2511
encapsulate clay platelets but most of them resulted in the so-
called armored latex particles, where platelets were located at
the surface of the particle instead of being encapsulated. In this
study, negatively charged RAFT oligomers of various length of
about 10 to 20 units of monomers stabilized by carboxylic acid
groups were used to adsorb onto the surface of the cationic
hexagonal gibbsite platelets. The adsorption was driven by the
electrostatic interaction between the anionic carboxyl groups of
the RAFT oligomer and the cationic AlOH2+. The isoelectric
point of the gibbsite platelets is around pH 9, which provides
an ideal pH window for the encapsulation via the RAFT route.
The positive charge on the platelets originated from the
ionization of the surface AlOH− groups into AlOH2+ at pH
values below the isoelectric point.
Then polymerization was performed with the addition of
monomers, methyl methacrylate (MMA). The best encapsu-
lation was obtained by using RAFT oligomer BA5-co-AA10 with
a monomer feed composition of MMA:BA = 10:1. The
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resultant polymeric complex adopted the near-hexagonal shape
of the gibbsite demonstrating the growth of the polymer on the
Figure 7. (a) Chemical structure of DODAB and schematic of the
cationic DODAB vesicle. Adapted with permission from ref 59.
Copyright 2018 MDPI. (b) Cryo-TEM micrographs of vesicles
obtained after extrusion of 10 mM DODAB dispersion through triply
stacked 100 nm polycarbonate filters. Adapted with permission from
ref 24. Copyright 2010 American Chemical Society.
surface of the template (Figure 5c and d). It is worth noticing
that in the micrographs, the appearance of ellipsoidal
complexes were likely due to the orientation of the particles
in the sample. When the hexagonal platelets were visualized
from the side, the shape appeared to be ellipsoidal on a 2-
dimensional micrograph. Two variables were used to control
the thickness of the polymer, the amount of monomers fed
into the system and the ratio between butyl acrylate and acrylic
acid in the RAFT oligomer. Due to the greater hydrophobicity
of butyl acrylate compared to acrylic acid, when a RAFT
oligomer of BA7.5-co-AA10 was used, the higher overall
hydrophobicity promoted the formation of micelles in the
aqueous environment and hence less polymer was formed on
the surface of the gibbsite template, resulting in a thinner
polymeric membrane.26
The main limitation of the method is that the reaction
kinetics of selected monomers should be in the same order of
magnitude to effectively obtain RAFT control. However, the
study demonstrated a different approach to use a controlled
RAFT polymerization method in an aqueous medium to form
polymer/gibbsite nanocomposite latex particles. The use of the
aqueous medium made the method possible to be adapted in
pharmaceutics and demonstrated that RAFT polymerization
can serve as a mechanism to obtain products with controlled
size distribution and thickness. Performing an adsorption step
with RAFT short-chain oligomers made it possible to localize
the polymerization reaction through electrostatic interaction
on the surface of the template, which was the key to
successfully encapsulate the gibbsite and to ensure that the
final product adopted the shape of the template for a precise
control of morphology.
In another study, cubic and tetrahedral capsules using
cadmium carbonate (CdCO3) and tin sulfide (SnS) particles as
the template, and deposition of polymer via the layer-by-layer
method. The layer-by-layer method made use of the electro-
static attraction by opposite charges between the deposited
template and each layer.15 Cubic cadmium carbonate particles
(Figure 6a) were obtained by precipitation in solution of
cadmium nitrite containing urea; tetrahedral tin sulfide
particles (Figure 6b) were prepared by thermal decomposition
of tin chloride and elemental sulfur in oleylamine. Then three
bilayers of tannic acid (TA) and poly(N-vinylpyrrolidone)
(PVPON) were deposited on their surfaces. The inorganic
cores were then dissolved by shaking the particle dispersion in
different concentrations of HCl yielding hollow capsules
(Figure 6c and d). Hollow capsules with sharp edges are
difficult to construct due to high residual stresses.73 The layer-
by-layer method has been applied to prepare hollow non-
spherical capsules with various templates. It is believed that
hydrogen-bonded layer-by-layer shells have sufficient strength
to maintain the original cubic shape after the removal of the
core in multiple examples.74−76 In this study, to provide more
insight into the mechanical stability of the cubic and
tetrahedral hollow structures, a stress/strain model was
applied. It was found that in contrast to spherical micro-
capsules, cubic and tetrahedral hollow structures had improved
stability over a wide range of pH, which was attributed to the
built-in edges of the structure maintaining the capsule integrity
upon stress application. The study showed the possibility to
use layer-by-layer adsorption without a polymerization reaction
to produce nanocapsules of enhanced strength when
responding to external pressure modulation.56
To summarize, the successful coating of a layer of polymeric
material onto the surface of some nonspherical inorganic
particles first demonstrated the possibility to adopt the shape
of the template by the coated polymeric membranes. Inorganic
templates are usually stable during the polymerization
reactions and the nonspherical morphologies are maintained
for the polymeric membrane to deposit. A driving force is

Figure 8. Cryo-TEM micrographs of (a) structures after polymerization of styrene with extruded DODAB vesicles. Phase separation occurred and
gave rise to parachute-shaped vesicle-polymer complex. Small polymeric spheres were linked to the vesicle bilayers. The scale bar corresponds to
100 nm. Adapted with permission from ref 57. Copyright 1997 American Chemical Society. (b) More spherical vesicles and polymeric particles
after polymerization of styrene with DODAB vesicles at 60 °C. Arrows point to the isolated polymer latex particles (x) fully separated from the
“parental” vesicle. The scale bar corresponds to 100 nm. (c) Duplicated parachute morphologies with the larger shell containing the smaller one
after thermally induced polymerization of styrene with DODAB vesicles at 60 °C using the water-soluble initiator V50. The scale bar corresponds
to 100 nm. Adapted with permission from ref 58. Copyright 2000 American Chemical Society.

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Chemistry of Materials
Figure 9. Cryo-TEM micrograph of polymerization of DODAB
vesicles with methyl methacrylate (MMA). Parachute-shaped vesicle-
polymer complex and protrusion-shaped nanocapsules were formed
without RAFT control. Arrows show the difference between the two
types of structures with (protrusion-shaped) and without (parachute-
shaped) a layer of polymeric shell on the surface of the vesicles.
Adapted with permission from ref 59. Copyright 2018 MDPI.
needed to attract the polymerization reaction onto the surface
of the inorganic template such as the capture of the monomer
radical by the vinyl functional group of the template in the first
example and the electrostatic attraction in the second and third
examples. A major challenge in producing nonspherical
nanocapsules is that once the synthetic template is removed,
the polymeric membrane can revert back to a spherical shape
which is believed to be more energetically favored in most
cases. Therefore, comprehensive engineering is required to
optimize the system before obtaining stable nonspherical
nanocapsules. After the removal of templates, either the cross-
Figure 10. Percentage of protrusion-shaped nanocapsules varied when different amount of different cross-linkers with various hydrophilicity were
used, including ethylene glycol dimethacrylate (EGDMA), (EG)10DA, and ethylene glycol diacrylate (EGDA). Adapted with permission from ref
43. Copyright 2018 Elsevier.
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linking of the polymeric shell or the intermolecular interaction
(in the third example of layer-by-layer coating) prevented the
polymeric capsules from reverting to spherical morphologies.
The thickness of the shell can also be modified simply by
feeding various amount of monomers, cofeeding a cross-linker
or a monomer with similar structures to further help reduce
the reversion of shape after template removal. In the context of
drug delivery, using inorganic templates limits the application
of the final polymeric nanocapsules because in the template
removal step, to obtain an internal cavity, strong solvents acids
are used and the harsh conditions are likely to compromise the
activity of pharmaceutical actives.
3.2. Parachute-Shaped Vesicle-Polymer Complex,
Protrusion-Shaped Nanocapsules, and Nanobottles.
To improve the applicability of nanocapsules in drug delivery,
soft vesicles made by surfactant molecules or phospholipids
(liposomes) were used in preference to inorganic templates.
Some amphiphilic molecules, such as dimethyldioctadecyl
ammonium bromide (DODAB),24,32 sodium dodecyl benze-
nesulfonate (SDBS),77 or phospholipid including egg phos-
phatidylcholine (egg-PC),22 have been used to form nano-
capsules via free radical or reversible-deactivation radical
polymerization. They were applied in the fields of drug
delivery, cosmetics, personal care, and coating.16,49,78 Com-
pared to the inorganic hard templates summarized in the
previous section, soft templates are much more biologically
applicable and do not require a template removal step. They
are also referred to as nonsacrificial templates, which avoids the
degradation of pharmaceutical actives during hard template
removal.
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Figure 11. Schematic of the concept of a nanobottle. The active
ingredient is retained by the highly impermeable “wall” part. The
small “lid” part can respond to an external trigger (light, temperature,
pH, ionic strength etc.) to become more permeable and release the
cargo. The lid can be closed again reversibly. Adapted with permission
from ref 61. Copyright 2016 Taylor & Francis Group.
In some examples of using a soft template such as the use of
DODAB vesicles to form hollow spherical nanocapsules, the
DODAB template layer was too thin to be observed in the
micrographs of the final nanocapsules. However, because of the
soft nature of such a template, it could be challenging to
maintain the desired morphologies. In this section, the use of
soft templates to prepare parachute-shaped vesicle-polymer
Figure 12. (a) Schematic illustration of the procedure used to seal the
holes on the surfaces of hollow particles with corks made of a phase-
change material (PCM), (b) SEM, and (c) TEM images showing the
particles corked with 1-tetradecanol. Reprinted with permission from
ref 62. Copyright 2013 John Wiley and Sons.
complex and protrusion-shaped nanocapsules are introduced.
The application of using a soft template in drug delivery has
not yet been researched in-depth, so based on currently
available research in the literature, two future perspectives are
introduced, namely, the nanobottles and encapsulation of drug
nanocrystal to serve as an elongated drug-loaded template.
3.2.1. Vesicle Template Formed by DODAB. Dimethyl-
dioctadecyl ammonium bromide (DODAB) (Figure 7a) is a
double-chain cationic surfactant. Because of its cost-effective-
ness and biological compatibility, it was used to form surfactant
vesicles as the template to prepare protrusion-shape nano-
capsules (Figure 7a). Unilamellar vesicles were obtained by
extruding DODAB dispersions through triply stacked 200 nm
polycarbonate membranes (Figure 7b). Liposomes made from
natural phospholipids are typically spherical; however, the
DODAB vesicles contained sharp corners and edges, which
was likely to be caused by the charged headgroup of DODAB
molecules. The rigid bilayer created packing constraints and
the charge repulsion results in the “pointy” edges. This is a
common phenomenon of charged surfactant vesicles.24,79 A
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recent publication confirmed the influence of ion content in
water (NaCl, NaBr, and LiCl) used for DODAB vesicles. They
affected the size distribution and morphology of the vesicles,
which in turn affects the suitability of those vesicles to be used
for polymerization, the final size and morphology of resulting
nanocapsules. Therefore, in preparation of vesicle templates for
nanocapsules, the quality of water and control over ionic
strength is important.80
3.2.2. Parachute Vesicle-Polymer Complex and Protru-
sion-Shaped Nanocapsules. A parachute-shaped vesicle-
polymer complex was first obtained prior to the preparation
of protrusion-shaped nanocapsules via free radical photo-
polymerization of styrene with DODAB vesicles as the
template. If polymerization occurs on the surface of the
vesicles, a layer of polymeric shell will form. If polymerization
occurs in the reaction media or phase-separates from the
vesicles, solid polymeric balls will form to minimize the contact
with the media. In this case, nearly all observed “parachute-
shaped” complexes possessed one solid polymeric particle
attached to the side of the vesicle template (Figure 8a).
Further investigations found that the bilayer properties of the
DODAB vesicle after polymerization were unchanged, mean-
ing that there was no polymeric material coated on the surface
of the vesicles and the polystyrene beads had completely
phase-separated from the DODAB vesicles. However, the
polymer beads were still attached to the parent vesicles. It was
concluded that when DODAB was used as the material for the
vesicle template, styrene as the monomers and radical
polymerization was performed, regardless of the type (photo-
or thermal-) of initiation, type of initiators, or temperature of
polymerization, parachute-shaped vesicle-polymer complex
could be formed.57 Further investigation has shown that
reaction temperature (increased from 25 to 60 °C) made both
the solid polymer particles and the vesicle bilayer more
spherical (Figure 8b). The use of a water-soluble initiator V50
produced duplicated parachute morphologies with the larger
shell containing the smaller one (Figure 8c).58 The studies
demonstrated the importance of understanding and applying
more complete knowledge of the relationships between
polymeric structures of desired shapes and the reaction
conditions, which was governed by fundamental thermody-
namic control.
The formation of protrusion-shaped nanocapsules further
demonstrated the importance of the reaction kinetics and the
balance between the hydrophobicity of the vesicle and the
formed polymer on the resultant morphology. Visually
protrusion-shaped nanocapsules and vesicle-polymer com-
plexes appear to be very similar in the fact that they both
have two compartments, a vesicle part and a solid polymeric
part. However, around the vesicle part of a protrusion-shaped
nanocapsule, there appeared to be a layer of polymer wrapped
around the vesicle and connected to the solid polymeric part
on the side (Figure 9). To form the parachute-shaped vesicle-
polymer complex, polymerization occurred in the bilayer
leading to a complete phase separation. To form the
protrusion-shaped nanocapsules, polymerization was directed
onto the surface of the DODAB vesicle by using a short chain
oligomer incorporating a RAFT agent. RAFT control was
introduced to form homogeneous spherical hollow nano-
capsules via the vesicle-templated method with the adsorption
of short chain oligomer on the surface of the vesicles prior to
the addition of initiators and monomers. However, when
methyl methacrylate (MMA) was used as the monomer
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Figure 13. (a) Standard TEM image and (b) the enlarged image
showing the ciprofloxacin nanocrystals. Scale bars represent 500 and
200 nm, respectively. (c) Cryo-TEM image showing the lipid bilayer
made of hydrogenated soy phosphatidylcholine (HSPC) and
cholesterol, which encapsulates the drug nanocrystal inside. Scale
bar represents 200 nm. Adapted with permission from ref 85.
Copyright 2019 American Chemical Society. (d) Cryo-TEM image of
Doxil, which is ammonium sulfate loaded doxorubicin pegylated
liposomes; a linear rod-shaped doxorubicin sulfate nanocrystal is
observed inside each liposome. Adapted with permission from ref 83.
Copyright 2012 Elsevier. (e) Cryo-TEM image of doxorubicin HCl
loaded into liposomes using a citrate gradient showing circular and U-
shaped nanocrystals. Adapted with permission from ref 88. Copyright
1998 Elsevier.
instead of methyl acrylate (MA), a polymerization without
RAFT control occurred resulting in the formation of the solid
polymeric particles on the side.59,60 To be capable of designing
nonspherical nanocapsules of desired morphologies, a deep
Figure 14. (a) Schematic of the formation of core−shell like and nonspherical seed particles by adding MMA/AIBN and St/AIBN. (b) TEM
micrograph of P(St/DVB/AA)@PS particles. (c) TEM images of final particles prepared from P(St/DVB/AA)@PS seeds with MMA:DVB = 1:2.
Adapted with permission from ref 64. Copyright 2017 Elsevier.
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understanding of the mechanistic role of various reagents on
the reaction kinetics and properties of the formed polymer is
needed.
A quantitative cryo-TEM study showed that increasing the
amount of cross-linker, ethylene glycol dimethacrylate
(EGDMA), increased the quantity of protrusion-shaped
nanocapsules. The use of more hydrophilic cross-linkers,
such as (EG)10DA and ethylene glycol diacrylate (EGDA)
produced less protrusion-shaped structures (Figure 10).43 The
example demonstrated that by altering the balance between the
kinetics of polymerization and the rate of phase separation, the
morphology of the final nanocapsules was controlled.
3.2.3. Future Perspective 1: The Concept of Nanobottles.
Morphologically, a protrusion-shaped nanocapsule with hollow
main body and an attached solid ball is similar to that of a
bottle with a lid. On the one hand, it inspired the idea of using
different materials for the “wall” and the “lid”, respectively, to
retain and deliver pharmaceutical actives of various phys-
icochemical properties in one carrier. On the other hand, a
bottle can be “open” or “closed” using a stimulus, similar to the
concept of on-demand drug delivery. For an on-demand drug
delivery carrier, drug permeability and release can be different
upon various external stimulation through distinct materials
used. Nanobottles are morphologically similar to protrusion-
shaped nanocapsules, with a cross-linked impermeable “wall”
as the main body and a small connected stimuli-responsive
“lid” that can be opened and closed reversibly under stimuli
control (Figure 11).61 In this way, the nonspecific leakage of
active ingredients through the body of the carrier is minimized
and the release of active ingredients can be controlled via
responsive mechanisms. One study of polymer nanobottles
used polystyrene as the material for the “wall” and a
temperature-responsive material as the phase-change material
(PCM), 1-tetradecanol, as the “lid”. The “wall” was 200 nm-
thick and very impermeable. The “lid” was capable of
reversible, solid−liquid transition in response to temperature.
When heated above 38−39 °C, the encapsulated active
ingredients were released (Figure 12).62 A recently published
review summarizes the fabrication, functionalization and
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application of nanobottles.63 Preparing nanobottles with
distinct materials to achieve impermeability of the wall and
reversible responsiveness of the lid is a challenge. However, the
existent development of nanobottles demonstrates the versatile
applications of nonspherical nanocapsules not only in cellular
interactions, which makes use of the physical morphology but
also in tuning the intrinsic properties of different parts of the
nanocapsules for a controlled and responsive release of active
ingredients.
3.2.4. Future Perspective 2: Elongated Vesicle Template
Containing Drug Nanocrystals. Another future perspective of
using a soft vesicle template was to use nonspherical liposome
or surfactant template to form nonspherical nanocapsules.
Previously, nonspherical nanocapsules were prepared with
inorganic templates because of their ability to maintain the
elongated shape throughout the polymerization reaction.
However, the template occupies the internal cavity of the
formed nanocapsule and its removal limits the potential to
encapsulate pharmaceutical actives prior to polymerization.
Therefore, soft vesicle templates were preferred with the
internal cavity to encapsulate drugs and the potential to bypass
the removal of templates. To direct the polymerization
reaction onto the surface of the vesicle template so that the
polymeric layer “wrap” around the template, Rusli et al.
performed an adsorption step prior to polymerization. A
vesicle prepared using a positively charged surfactant, DODAB,
was used as the template, which was then added to a solution
of RAFT oligomer, a type of short-chain oligomer made with
an incorporated RAFT agent, acrylic acid, and butyl acrylate.
The ionization of the carboxyl group provided a negative
charge, which drives the RAFT oligomer to attach onto the
positively charged DODAB vesicle surface. Spherical hollow
nanocapsules were successfully prepared with a homogeneous
layer of polymer formed by RAFT polymerization. The
example made it possible to attach a polymeric layer directly
onto the surface of soft vesicle templates instead of having the
polymerization in the bulk solution forming polymeric solids.59
When it comes to soft vesicular templates, two types of
templates have been explored−surfactant vesicles and lip-
osomes. Liposomes are prepared by self-assembly of
phospholipids, which is a natural component of cell
membranes containing a glycerophosphate group while
surfactants are synthetic molecules. To compare vesicles
prepared using phospholipids and surfactants, there are two
benefits of using liposomes over surfactant vesicles. First, the
successful formation of stable DODAB vesicles relies on using
a low concentration of NaCl solution, 0.5 mM, upon
hydration,59 which limits its application in drug delivery
where biological fluids possess a much higher ionic strength.
Second, liposomes have been widely used in encapsulating a
great variety of drugs of different physical states, including drug
nanocrystals.81
In the context of this review, liposomes containing drug
nanocrystals are of great interest because the growth of the
drug nanocrystals inside liposomes elongated the vesicles and
created a nonspherical morphology. The drug was first
encapsulated in solution, usually by an active loading method
to achieve high encapsulation efficiency, and then the drug
nanocrystal was formed inside liposomes. Depending on the
method of encapsulation and the physiochemical properties
and chemical structure of the drug, the encapsulated drug may
exist as a crystalline precipitate, amorphous precipitate, or in a
supersaturated or subsaturated solution.82 For example, some
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Figure 15. (a) TEM images of anisotropic polymer particles with
silica particles made with MMA and St in the first and second
polymerizations and (b) TEM image of anisotropic hollow particles
obtained by immersing anisotropic composite particles into hydro-
fluoric acid for a week. Adapted with permission from ref 65.
Copyright 2008 John Wiley and Sons.
drugs, such as doxorubicin, formed nanocrystals inside
PEGylated liposomes without further processing,83 while
some other drugs, such as ciprofloxacin, required an extra
step of freeze−thawing to crystallize inside liposomes.81 The
freeze−thawing process involves the freezing of the drug-
loaded liposomes in the presence of a cryo-preserver, such as
sucrose, usually in liquid nitrogen and then thawing them at
room temperature or some controlled temperature environ-
ment. It was proved that when preparing liposomes using
ciprofloxacin HCl that ciprofloxacin base was formed in the
thawing process84 with the formed ice crystal acting as the
crystallization nuclei. Aspect ratio of the liposomes was
controlled by formulating the lipid bilayer with cholesterol to
modify the rigidity of the bilayer. In addition to ciprofloxacin
(Figure 13a−c)85,86 and doxorubicin (Figure 13d−e), a range
of therapeutic actives are able to form nanocrystals inside
liposomes and elongated the liposomes. Different shapes and
aspect ratio of nanocrystals87 are also obtained, depending on
the coupling of different salts in the case of doxorubicin sulfate
(Figure 13d) and doxorubicin HCl (Figure 13e).82,83,88 For a
certain shape of nanocrystals, such as the elongated shape, the
aspect ratio could also be adjusted by the addition of different
concentrations of sucrose, amount of cholesterol, the drug to
lipid ratio, or by using different phospholipids with different
phase transition temperatures.87
Being able to preload drugs in nonspherical template
combines the benefits of the low systematic excretion of the
elongated morphology and the slower release of drugs from the
thick polymeric membrane. DODAB vesicles made use of the
positive charge on the surface to drive adsorption of negatively
charged RAFT oligomers. In the case of liposomes, surface
charge or functional groups could be used as the driving force.
For example, elongated liposomes encapsulating ciprofloxacin
nanocrystals have been successfully prepared with azide
groups, which could potentially be used to drive RAFT
oligomers with the complementary chemical groups onto the
surface.84
In this section, the more biocompatible approach using soft
vesicular templates prepared from surfactants or phospholipids
was presented. The vesicular feature of these templates allowed
potential encapsulation of drugs. Previous research in
encapsulating drugs in different physical state especially in
the form of nanocrystals stretching the liposomes provided an
elongated template. The successful functionalization of these
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Figure 16. (a) Schematic of the preparation of the thermosensitive hollow Janus dumbbells. The template is prepared in steps I and II and
subsequently covered by a shell of PNIPAM. Dissolution of the template in step IV leads to hollow Janus dumbbells. (b) Cryo-TEM micrograph of
a PS dumbbell core particle showing one lobe with PS sphere only and the other lobe with MPS coated PS sphere indicating a core−shell structure.
(c) Cryo-TEM micrograph of thermosensitive hollow Janus dumbbell-shaped microgels at 20 °C in water. The image proves the Janus character of
the particles showing a hollow side of the microgel (light gray) and partially hollow part (dark gray) composed of PNIPAM shell and PS-co-PMPS
copolymer layer. Image provided courtesy of Matthias Ballauff.

Figure 17. (a) Schematic of the preparations of elephant trunk-like and acorn-like hollow Janus particles. (b) TEM micrographs of the solid Janus
particles, (c) elephant-trunk-like hollow Janus particles at low (×5k) and (c′) high (×15k) magnifications, and (d) acorn-like hollow Janus particles
at low (×5k) and (d′) high (×15k) magnifications. Adapted with permission from ref 67. Copyright 2014 American Chemical Society.

liposomes with “click” chemistry groups provided a promising
driving force to locate the formation of polymers onto the
surface of the template. Their biocompatibility allowed the
templates to be embedded in the final nanocapsules, which
omits the template removal step and provides a much better
opportunity to maintain the bioactivity of drugs. However,
maintaining the nonspherical shape was more challenging than
that of inorganic templates and therefore, kinetics of
polymerization reactions with different monomers and the
use of cross-linker were investigated. This was reflected on the
formation of polymer-vesicle complex and protrusion-shaped
structure where different polymerization kinetics, combined
with the hydrophobicity of the formed polymer, played a
crucial role in the separation of the “protruded” part and the
thickness of the shell. This morphology led to the idea of
nanobottles where the “wall” part is made of inert materials to
ensure encapsulation of actives at all times and the “lid” part is
made of stimuli-responsive materials, which becomes leaky to
release the cargo upon stimulation. Although challenges still
remain to ensure the inertness of the “wall” material and the
ability of the “lid” to “open” and “close” reversibly, the use of
soft templates and development of nanobottles expanded the
2517
application of nonspherical nanocapsules from a simple
asymmetrical system to a multifunctional stimuli-responsive
drug delivery system.
3.3. Dumbbell-Shaped and Snowman-Shaped Poly-
meric Nanocapsules. Polymeric nanocapsules with more
than one internal cavity attracted a lot of interest in research
because of the potential to incorporate multiple properties and
functionalities in one nanocapsule. Dumbbell-shaped and
snowman-shaped polymeric nanocapsules are the most
investigated nanocapsules with two partially fused internal
cavities. Some of them were made with a homogeneous layer
of polymer while others were made of two or more materials in
different parts. One important class of dumbbell- and
snowman-shaped nanocapsules are the hollow Janus nano-
particles. Janus nanoparticles are anisotropic particles with two
or more lobes with distinct physical, chemical, optical, or
surface properties.89,90 In this section, the formation of
dumbbell-shaped and snowman-shaped polymeric nanocap-
sules including Janus-type particles will be discussed.
3.3.1. Dumbbell- and Snowman-Shaped Nanocapsules.
Dumbbell-shaped latex particles with a void on one side were
prepared via seeded emulsion polymerization. To prepare the
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Figure 18. (a) Structures of the block copolymers PEG43-b-P(NIPAM21-co-PDMI9) with and without the targeting peptide. (b) Schematic of
polymersomes prepared using a solvent-switch method. Copolymer without any peptide ligand and copolymer with high or low peptide densities
were dissolved in THF (in red). Addition of water (in blue) induces self-assembly of the polymer. Different ratios of polymers with and without
peptide ligands in “step 1” are key in tuning the ligand density of the polymersomes, while the increasing volume of THF corresponds to an
increased sizes of polymersomes. (c) TEM images of the large and (d) small low-ligand density polymersomes of varying peptide ligand-density
(expressed in percentage). Scale bars: 200 nm. Adapted with permission from ref 68. Copyright 2020 John Wiley and Sons.

nonspherical seed, styrene (St), or methyl methacrylate
(MMA) mixed with azobis(isobutyronitrile) (AIBN) into a
cross-linked spherical poly(styrene/divinylbenzene/acrylic
acid) P(St/DVB/AA) particle was swelled and polymerized
(Figure 14a). One lobe was made of cross-linked P(St/DVB/
AA) and the other lobe was made of PS (Figure 14b). To
prepare the nonspherical hollow latex particle with a void on
the PS side, polymerization was performed with MMA/DVB/
AA monomers at a different ratio. The seed removal step was
not needed because polymerization happened on the surface of
the seed, followed by a phase separation between the PS seed
and the double-layered shell. As polymerization proceeded, the
non-cross-linked PS and DVB moved from the inner part
where the polymerization occurred to compensate for the
consumed monomer. Consequently, the void was expanded
(Figure 14c). The double-layered shell composed of PS inner
shell and P(DVB/MMA/AA) outer shell prevented the
collapse of the hollow void. The one-sided hollow feature of
the dumbbell-shaped particle and the rigid shell made them
2518
useful in coating applications with specific optical properties
and sufficient mechanical durability.64 The study not only
provided a good example of dumbbell-shaped particles with a
void only in one of the two lobes, but also presented a method
to create an interior cavity without the need to remove the
original template.
In another study, snowman-shaped nanocapsules with one
void was prepared with the presence of a silica particle which
was then dissolved with hydrofluoric acid to obtain the interior
cavity. They were prepared with a two-step emulsion
polymerization. In the first step, seed polymer particles (Figure
15a) were prepared in the presence of the reactive silane
coupling agent 3-methacryloxypropyltrimethoxysilane
(MPTMS) in water. In the second step, polymerization was
performed in induced anisotropic protrusion of polymer from
the seed particles. The study presented the possibility to apply
anisotropic polymer particles containing silica particles to
prepare anisotropic hollow nanocapsules with one void (Figure
15b). It also provided an option to adjust the size ratio of the
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Figure 19. TEM (left) and cryo-TEM (right) images of faceted polymersomes prepared using the solvent-switch method at (a and b) 65% and (c
and d) 70% THF/water. Higher magnification images are shown inset for clarity. Adapted with permission from ref 70. Copyright 2019 Royal
Society of Chemistry.
two lobes to make snowman-shaped nanocapsules. The optical
anisotropic scattering properties of the particles enabled their
use as photonic materials.65
3.3.2. Janus Nanocapsules. Janus nanoparticles of various
shapes including snowman,91 disk,92 dumbbell,93 and cylin-
der94 have been prepared using different types of materials,
such as lipids, dendrimers, inorganic material, and polymers.
Because of the spatial discrimination of two or more properties
in one particle, Janus nanoparticles have a wide range of
applications in pharmaceutics, coating, and colloidal chem-
istry.95 Polymers have been used extensively to prepare Janus
nanoparticles due to the availability of easily modifiable
polymeric groups and the possibility to incorporate stimuli-
responsive properties. Janus nanoparticles can be mainly
divided into solid nanoparticles and hollow nanocapsules.
Only hollow nanocapsules will be presented due to the
presence of the internal cavity, which is within the scope of this
review. Seeded emulsion polymerization has been the most
practical method to prepare nonspherical hollow Janus
nanocapsules not only as dumbbell66 and snowman shaped
but also with an “elephant trunk” shape.67
The encapsulation of a wide range of drugs into Janus
nanoparticles, such as ketoprofen,96 doxorubicin,97 and some
macromolecules, such as proteins98 or DNA,99 has been
achieved by using different encapsulation methods including
microfluidics, solvent incorporation and chemical cross-linking.
In the study by Khan et al., two model drugs with distinct
physicochemical properties, ketoprofen and sodium fluores-
cein, were first incorporated with two different types of
monomer, acrylamide and methyl acrylate, followed by rapid
synthesis via a side-by-side microfluidic device. Janus particles
were formed by UV-assisted free radical polymerization.
Factors, such as flow rates of the microfluidic device, monomer
composition of the two compartments, nature and concen-
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tration of the surfactant, outlet tube diameter, and UV
intensity, were controlled to obtain different particle shapes
ranging from core−shell to bicompartmental particles. Both
drugs were released following a diffusion type mechanism.96 In
the case of Janus capsules made by Wu et al. for a light-
triggered release of doxorubicin (DOX), DOX was encapsu-
lated using an organic solvent after the formation of the Janus
capsules. The method is based on the organic solvent partially
breaking the electrostatic attraction between the two polymeric
materials of the capsule, poly(styrenesulfonate) sodium salt
(PSS) and poly(allylamine hydrochloride) (PAH), which
largely increased the permeability to allow DOX to be
encapsulated upon mixing.100 The alcohol was then removed
by centrifugation. The successful encapsulation of DOX was
confirmed by UV−visible spectroscopy.97 In the case of
delivering proteins, four different types of proteins were
conjugated to the two sides of Janus nanoparticles selectively
by a gold deposition method. In brief, the shell on either side
of the Janus nanoparticles was selectively modified via an
amino-alkanethiol reagent or thiol-PEG-biotin to cross-link
proteins.98 The silica particles were functionalized with (3-
aminopropyl)triethoxysilane (APTES) or bovine serum
albumin (BSA), followed by functionalization of the gold
hemisphere with amino-alkanethiol solution and attachment of
natural human fibronectin (FN).98 Drug entrapment is
possible with Janus nanoparticles and methods need to be
selected according to the size, property, location of entrapment
of the drug and the types of the material used. However, the
below presented examples of nonspherical Janus nanocapsules
focus more on the morphology and dual chemical functionality
of the Janus nanocapsules instead of drug entrapment.
Thermosensitive hollow Janus nanocapsules with a dumb-
bell-shape were successfully prepared using dumbbell-shaped
microgels as templates.66 They consist of two partially fused
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Figure 20. TEM images and proposed self-assembly pathways for the different morphologies observed. (a−e) TEM images. The insets in panels a−
c are higher magnification TEM images. (f−j) Cryo-TEM images. The insets in panels g−j are higher magnification cryo-TEM images and intensity
plot profiles used to estimate membrane thickness of individual polymersome morphology. (k−o) Schematic representations of the micelle/
polymersome structures formed. The black arrows in panels l−o indicate the direction of membrane tension. Scale bars in a−j are 100, 200, 500,
2000, 2000, 50, 100, 100, 300, and 1000 nm, respectively. Scale bars inset in a−c are 50, 100, and 100 nm, respectively. Adapted with permission
from ref 71. Copyright 2017 Springer Nature.

hollow spheres. One sphere has a shell completely made of
poly(N-isopropylacrylamide) (PNIPAM) and the other one
has a hybrid shell made of a poly(styrene-co-3-
(trimethoxysilyl)propyl methacrylate) (PS-co-PMPS) layer
covered by PNIPAM. To prepare these hollow Janus
dumbbells, polystyrene (PS) spheres were prepared as seeds
and then coated by a copolymer layer of styrene and 3-
(trimethoxysilyl) propyl methacrylate (MPS). Then the
resultant core−shell particles were swollen with free styrene
and polymerized, which provided the dumbbell-shaped core.
Cross-linked PNIPAM was attached onto the surface of the
dumbbell-shaped core via seeded emulsion polymerization.
Lastly, the PS core was dissolved in tetrahydrofuran (THF) for
core removal leaving the Janus dumbbells hollow (Figure 16a).
Cryo-TEM micrographs of the dumbbell-shaped seed demon-
strated that it consists of two partially fused spheres−one side
with a homogeneous PS sphere and the other side with a PS-
co-PMPS core−shell structure (Figure 16b). After dissolving
the PS core, the Janus character was obvious in the cryo-TEM
micrograph (Figure 16c) with one lobe having a visually lighter
hollow interior because the original template for that side was
made of PS alone. The other lobe was partially hollow due to
the insolubility of the PS-co-PMPS core−shell template in
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THF. The hollow nature of the hybrid shell (darker gray color
in Figure 16c) was hard to visualize in the cryo-TEM
micrograph. However, the gray scale profile along the center
of the particle indicated clearly a decrease in contrast toward
the center, which proved both lobes to be hollow. The
thermosensitivity of the Janus hollow dumbbells was studied
using dynamic light scattering (DLS) and depolarized dynamic
light scattering (DDLS), which showed that the shell thickness
decreased from 140 nm at 10 °C to 50 nm at 45 °C. As a
result, the aspect ratio of the particles was also tunable through
the control of temperature. The asymmetry in thermosensi-
tivity of the two different lobes of the Janus dumbbell-shaped
nanocapsules, therefore, extended the applications of non-
spherical nanocapsules using more modifiable materials as
carriers for drugs, nanoreactors, or catalysts.
Hollow Janus particles with the so-called elephant trunk-like
and acorn-like shapes (variations on the dumbbell-shape),
were prepared by seeded emulsion polymerization. In this case,
the hollow structure was obtained directly during the
preparation by a one-step swelling method in the presence of
toluene (Figure 17a). The seeds were made of a polystyrene
core with SiO2 shell (PS@SiO2) or 3-(trimethoxysilyl)propyl
methacrylate-SiO2 (PS@MPS-SiO2) core−shell particles (Fig-
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ure 17b) for the elephant trunk-like shape (Figure 17c) and
the acorn-like shape (Figure 17d), respectively. In the aqueous
phase of the emulsion, PS@SiO2 and PS@MPS-SiO2 cores
were mixed with sodium dodecyl sulfate (SDS). In the
nonaqueous phase of the emulsion, the monomers, the
swelling agents and the initiator were added at a specific
ratio to carry out the polymerizations. Using toluene as the
swelling agent was important for the formation of the hollow
structures. When PS@MPS-SiO2 was used instead of PS@
SiO2, the hydrophobicity of the seed particles was enhanced,
the spreading coefficient of the monomer was increased, and
thus the formed polymer layer tend to “spread” on the surface
of seed particles. As a result, the elephant trunk-like shape was
changed to an acorn-like shape. Factors influencing the
morphology control were investigated including the amount
of surfactant and the concentrations of cross-linker. It was
found that the balance between hydrophilicity and hydro-
phobicity and the efficiency of phase separation were crucial
for the preparation with tailored shapes. These amphiphilic
particles were used to stabilize emulsions.67
The study provided the opportunity to form hollow
asymmetrical Janus particles without the need to remove the
core by using strong solvents, which made it applicable to be
used to encapsulate solvent-sensitive cargo such as most
pharmaceutical, biological active, personal-care compounds.
However, the presence of the internal cavity was not clear in
the shown TEM images and the differences between the so-
called elephant trunk and acorn-like structure were not clearly
apparent. Comparing the aforementioned examples, a
template-removal step is still essential to successfully prepare
the hollow voids. It is also recommended that cryo-TEM can
be used instead of TEM to avoid structural change of the
nanocapsules upon dehydration, such as polymeric wall
squashing together destroying the internal cavity in the images.
Dual- or multicompartmental nanocapsules in dumbbell-,
snowman-, or other shapes, including Janus nanocapsules
further widen the application and versatility of nonspherical
nanocapsules. To obtain the partially fused multicompartmen-
tal nanocapsules, templates, such as silica particles, are usually
first modified with a layer of polymer to form a core−shell
structure. Then making use of different kinetics of swelling and
polymerization for the core and the shell, protrusion of the
second compartment was formed. By adjusting the rates of
polymerization and the use of various materials, the volume
ratio between the two compartments can be adjusted. The
original templates can then be fully or partially removed
leaving the nanocapsules with one or two voids. Because of the
use of the modified core−shell template, the final materials
forming the two shells are usually different (Janus nano-
capsules) providing dual functionality. Dual- or multicompart-
mental nanocapsules serve as an example of not only making
use of the nonspherical feature in morphology but also in the
distinct intrinsic functionalities.
3.4. Nonspherical Self-Assembled Polymersomes. As
defined earlier, nonspherical polymeric nanoparticles consist of
an interior compartment, including polymeric membrane
bound by covalent bonds (sections 3.1−3.3) and polymer-
somes formed by self-assembly (section 3.4), are within the
scope of this review. Nonspherical polymersomes have been
reviewed previously,101 and therefore, this is not an extensive
review for nonspherical polymersomes. However, it is
significant to present the formation of nonspherical polymer-
somes because the mechanism of formation is different from
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that of a covalently cross-linked or cross-linked polymeric
membrane. For covalently cross-linked nanocapsules, the
construction of the nonspherical shape relied either on the
Figure 21. Cryo-TEM images following the shape transformation of
PEG22-PDLLA45 from (a) spherical polymersomes into elongated
nanotubes under the influence of osmotic pressure by dialysis, as a
function of [NaCl]; (b) 5, (c) 10, and (d) 50 mM. All scale bars =
500 nm. Adapted with permission from ref 69. Copyright 2016
American Chemical Society.
shape of the template (inorganic or surfactant vesicles), or the
different kinetics of polymerization on different parts of the
nanocapsules (such as in the case of the formation of
dumbbell-shaped nanocapsules via the swelling of polystyrene
in64). For self-assembled polymersomes, the formation of
nonspherical morphology relies on the molecular interactions
within the structure responding to an environmental change,
such as the strength of the solvent or osmotic pressure, as will
be introduced in this section.
3.4.1. Polymersomes Formed via the Solvent-Switch
Method. The formation of ellipsoidal polymersomes via a
solvent-switch method using the block copolymer, PEG43-b-
P(NIPAM21-co-PDMI9) (Figure 18a) has been reported.68 The
perylene moiety in the polymer provided the hydrophobicity
and liquid crystallinity for the polymer to self-assemble into the
ellipsoidal shape (Figure 18a).102 The ellipsoidal shape was
formed through a solvent-switch method with tetrahydrofuran
(THF) as the good solvent and water as the antisolvent. After
obtaining a homogeneous solution of polymer in THF, water
was added to the solution triggering aggregation and self-
assembly. The THF was then evaporated for 16−20 h yielding
ellipsoidal polymersomes in water (Figure 18b). Mechanisti-
cally, the formation of an ellipsoidal shape instead of a
spherical shape relied on the aromatic perylene interactions.
Depending on the remaining volume of their preferred solvent,
THF, the tightness of the stacking between the aromatic
perylene groups varied which provided different strain in the
structure. Making use of the aromatic stacking mechanism, the
sizes of the polymersomes were tuned by adjusting the volume
of THF during formation to yield polymersomes with
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Table 5. Nonspherical Nanoparticles and Their Corresponding Cellular Uptake or Circulation Time upon Application in
Different Diseases

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Figure 22. Time-lapse video microscopy clips spanning 39 min of macrophages interacting with identical elliptical disk (ED) particles (major axis
14 μm, minor axis 3 μm) from two different orientations. (A) ED particle attaches to the cell along the major axis of the particle, and it is
internalized completely in 3 min. (B) ED attaches to the cell on its flat side and the cell spreads but does not internalize the particle. Continued
observation indicated that this particle was not internalized for >110 min. (Scale bars: 10 μm.) At least three cells were observed for each
orientation of each particle type and size. Similar results were observed in all repetitions. Adapted with permission from ref 51. Copyright 2006
National Academy of Sciences, U.S.A.

hydrodynamic diameters of approximately 80 (Figure 18c) and
200 nm (Figure 18d) when measured using dynamic light
scattering (DLS). The study demonstrated the possibility to
design ellipsoidal polymersomes with a functional peptide
moiety on the surface and tune the size of the final
polymersomes without losing the ellipsoidal morphology via
the solvent-switch method.
Varying shapes were then formed upon investigation of the
mechanism of formation by varying the THF:water ratio.
Faceted polymersomes were obtained with a similar block
copolymer, PEG43-b-P(NIPAM21-co-PDMI19), when the THF
content in water was between 65% and 70%. With a THF
content in water of 65%, most polymersomes appeared as a
hexagon-like shape with six edges and vertices (Figure 19a and
b). With a THF content of 70%, polymersomes were more
polydisperse with shapes, such as tetragons, pentagons, and
some irregular polyhedrons (Figure 19c and d). To investigate
the role of THF on the aromatic stacking of the polymer
molecules, samples were taken at different time points during
the evaporation process of THF and THF was doped in a
series of aqueous polymersome dispersion. It was found that,
when the doped amount of THF exceeded 5 vol%, the
intermolecular aromatic stacking interactions between individ-
ual perylene-bearing polymers within the polymersome
membrane was reduced, resulting in the loss of well-defined
faceted shapes yielding spherical polymersomes. The study
demonstrated that not only can polyhedral polymersomes be
prepared via the solvent-switch method by carefully controlling
the amount of THF and the duration of THF evaporation, but
could also be separated for purification at different time points
of the self-assembly process.70
The mechanistic role of the aromatic interactions on
directing the spatial arrangement within membranes to form
various morphologies and sizes was further demonstrated in
another study. Making use of the aromatic perylene
interactions, medium, large, giant ellipsoidal polymersomes
and tubular polymersomes were obtained with block
copolymer PEG43-b-P(NIPAM21-co-PDMI9) by using various
amounts of THF and water. As the amount of good solvent
THF was increased, the ellipsoidal polymersomes (Figure 20a
2523
and f) became larger in size but with a smaller aspect ratio
(more round). This was due to the decreased aromatic
stacking with the presence of higher amounts of good solvent
resulting in the transition from a tightly packed interdigitated
state for medium ellipsoidal polymersomes (Figure 20b and g)
to a loosely packed interdigitated state for large ellipsoidal
polymersomes (Figure 20c and h), then to a quasi-
interdigitated state for giant ellipsoidal polymersomes (Figure
20d and i). At a higher amount of THF, tubular-shaped
polymersomes (Figure 20e and j) were observed because the
single layer of copolymers reached the limit of being able to
bend or curve. Schematic representations of the monolayer or
bilayer structures of the micelle/polymersome demonstrated
the different molecular organization (Figure 20k−o). The
membrane thickness was doubled in tubular polymersomes
compared to ellipsoidal polymersomes confirming the
rearrangement of a bilayer in tubular polymersomes.71 In
Summary, the solvent-switch method was exploited to form
ellipsoidal, tubular, and faceted polymersomes of various sizes
in the nanoparticle range in a controlled manner. The aromatic
interactions built into the polymer backbone played a
mechanistic role to direct the spatial arrangement within
membranes and hence the formation of various morphologies
and sizes.
3.4.2. Polymersomes Formed via the Osmotic Pressure
Method. As an alternative to the solvent-switch method, an
out-of-equilibrium process selecting osmotic pressure and
permeability has been used to form stomatocytes and bowl-
shaped vesicles. Spherical polymersomes were first prepared in
an organic solvent/water mixture, followed by dialysis against
water to introduce osmotic deformation. Upon dialysis,
plasticizing solvents, THF, and 1,4-dioxane within the inner
compartment of the polymersomes diffused across the
membrane into the dialysis aqueous medium to maintain
osmotic equilibrium. The rapid loss of plasticizing solvent
causes the hydrophobic polystyrene domain to lose its fluidity
and recover the rigid state. Consequently, the reverse transport
of water from the dialysis medium into the polymersomes
generates an osmotic pressure difference, which folds the
polymersome membrane inward, leading to the formation of
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Figure 23. Average number of spherical and worm-like IgG-adsorbed
particles internalized in 22 h as determined by fluorescence
microscopy. Patterned bars indicate particle volumes equivalent to 3
μm spheres (p < 0.0000031) and solid bars indicate particle volumes
equivalent to 1 μm spheres (p < 0.000024). At least 50 cells were
counted for each sample. Adapted with permission from ref 108.
Copyright 2009 Springer Nature.
stomatocytes. Osmotic pressure and permeability initiated
controlled deflation, followed by reinflation of the polymer-
somes, leading to different shapes. The mechanism has been
fully understood by the bending energy which allowed future
design of polymersomes with various shapes with high
controllability and predictability.103−105 According to the
mechanism, one can extrapolate that the applicability of
osmotic pressure is dependent on the composition, perme-
ability of the polymersome membrane and the interaction
between the material and the plasticizing solvents.
In another study, it was found that not only the solvent
strength but also the ionic strength in water played a significant
role on the final morphologies of polymersomes (Figure 21).69
The phenomena was similar to that of the aforementioned
DODAB vesicles80 driven by the molecular arrangement in
response to a change of ion strength in water. The mechanism
of such a method in the formation of various morphologies can
be explained by the interplay between osmotic energy and
bending energy, which is determined by the chemical
composition, length of the polymer, temperature, solvent,
Figure 24. (a) Filomicelles with increasing lengths (L0) circulate longer after injection to rats up to a limiting length. The error bars show the
standard deviation for four or more rats. (b) Tumor size decreases for the longest filomicelles at the highest paclitaxel (TAX) dose. All data show
the average from four mice. The error bars show the standard deviation. Adapted with permission from ref 27. Copyright 2007 Springer Nature.
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and ion strength of the environment, as explained in detail in
the extensive review of nonspherical polymersomes.101
3.4.3. Polymersomes Formed via the Chemical Addition
or the Liquid-Crystalline Lattice Confinement Method.
Inspired by the interplay between the molecular arrangement
and environmental factors, an approach of chemical addition to
induce shape transformation was attempted. Azide handles
were added to the hydrophobic domain of the polymersome
membrane and a cross-linker containing the complementary
functional group, bicyclononyne (BCN), was added in excess.
Polymersomes were elongated to various extents as the molar
ratio between BCN to azide was varied, as a result from the
chemical kinetics of the strain-promoted alkyne−azide cyclo-
addition (SPAAC) leading to various levels of cross-linking.72
Addition of non-cross-linking chemicals such as cholesterol,
was also used to prepare tubular polymersomes.30 Some other
methods include the liquid-crystalline lattice confinement
strategy,106 which has also been used to induce shape
transformation of such a nontemplated self-assembled system.
Despite the variety of methods in the literature, which has been
reviewed extensively previously,101 the intrinsic mechanism of
formation for nonspherical self-assembled structures is the
molecular arrangement (such as stacking) and strain created in
response to environmental factors.
In this section, the formation of nonspherical polymersomes
in various shapes were presented. In previous examples of
covalently cross-linked nanocapsules, properties of various
templates, monomers, and kinetics of polymerization reaction
played an important role on the formation of nonspherical
morphologies. For self-assembled nonspherical morphologies,
the formation relied on the interaction between built-in
chemical groups in the polymeric backbone in response to
change of environmental conditions such as solvent strength
and osmotic pressure. The two types of polymeric shells have
different permeability and capability of encapsulation for
pharmaceutical actives as a result of the mechanism of
formation. Hence, the versatility allows a relevant method to
be chosen according to the specific need of each drug delivery
case.
In Summary, polymeric nanocapsules have been prepared in
a wide variety of morphologies and presented systematically
(Table 3). Understanding the mechanism, advantages and
disadvantages of the various preparation methods (Table 4) is
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Figure 25. (a) HeLa cell uptake of transferrin-coated rod-shaped gold nanoparticles smaller than 100 nm is lower than their spherical counterpart.
Adapted with permission from ref 115. Copyright 2006 American Chemical Society. (b) Uptake of STO, HeLa, SNB19 cells of gold nanoparticles
of different aspect ratios were different. Adapted with permission from ref 116. Copyright 2007 American Chemical Society.
Figure 26. Probing the mechanisms of cellular internalization by
using inhibitors of endocytosis. HeLa cells were incubated with the
indicated inhibitors as outlined in the graph. Percent internalization
was normalized to particle internalization in the absence of inhibitors.
Adapted with permission from ref 118. Copyright 2008 National
Academy of Sciences, U.S.A.
crucial to the successful construction of the nonspherical
morphology with an interior compartment. The deployment of
the nonspherical particles toward applications, such as
intracellular delivery, is discussed in the next section.
4. INTERACTIONS OF CELLS WITH NONSPHERICAL
POLYMERIC NANOPARTICLES
The major driving force for the study of nonspherical
nanoparticles as drug delivery systems is for the nonspherical
shape to control interactions with cells and tissues. The aim of
this section, therefore, is to introduce nonspherical polymeric
nanocapsules and their current and potential applications in
the drug delivery field. However, methods to prepare
nonspherical nanocapsules are still being fully established
and establishing the research into the encapsulation and
retention of activity of drugs is in its early stages. Therefore, to
discuss the role of nonspherical morphology on cellular
interactions, examples from the literature on nonspherical
polymeric nanoparticles are mostly discussed here (Table 5).
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The shape of nanoparticles is reported to impact on cellular
interactions, resulting in different circulation times when
injected into the bloodstream.111 The shape of the nano-
particles also influences accumulation in tumor tissues and
consequently their therapeutic effect. Using the interactions of
nanoparticles with macrophages as an example, nonspherical
polymeric particles were taken up at a slower rate than their
spherical counterparts.28,50 Polystyrene particles with spherical,
ellipsoidal, elliptical-disk shape, rectangular disks, and “UFO-
shape” were prepared to study the impact of shape on
interactions with alveolar macrophages. It was found that
macrophages were more efficient in internalizing spherical
particles than ellipsoidal particles. When the size of the cells
was close to the size of the ellipsoidal particles, whether or not
the cells were able to complete the phagocytosis process was
dependent on the orientation of the particles when they came
into contact (Figure 22). If the particles came into contact at
the major axis, cells were not able to complete phagocytosis.51
In another study, worm-like polystyrene particles with very
high aspect ratio (>20) were fabricated by stretching spherical
particles embedded in a polymer film. Those worms have a
volume ∼125 fold smaller than the average alveolar macro-
phage, but the length of the particles was approximately twice
the diameter of the cells. It was shown that worms exhibited
very little internalization compared to spheres (Figure 23) and
negligible phagocytosis by an alveolar macrophage cell line was
observed. The studies demonstrated that shape-induced
inhibition of phagocytosis of drug delivery particles is possible
and could be used to modify the plasma circulation time of
drug delivery carriers.108,109 Decuzzi et al. investigated
spherical, hemispherical, discoidal, and cylindrical shapes of
silica particles in mice and found that discoidal particles
showed lower liver accumulation.110 Nanoparticles with
irregular shapes, especially elongated shapes, are less prone
to phagocytosis, so longer circulation time and less
accumulation at nontumor organs were achieved. With the
great variety of literature on this topic performed by different
research groups, it is crucial to report adequate and quality
characterization of the materials to enable comparison across
studies, a point of recent intense discussion in the literature.112
Due to the asymmetrical shape slowing down the uptake of
particles by macrophages, an increased circulation time was
achieved.107,113 Filomicelles were self-assembled from diblock
copolymers and used to interact with phagocytes. In rodents,
they were found to persist in the circulation for up to 1 week
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Chem. Mater. 2022, 34, 2503−2530
Chemistry of Materials
after intravenous injection, which was about ten times longer
than their spherical counterparts. In a flow chamber with
immobilized phagocytes, long filomicelles flowed past the cells
but smaller micelles and vesicles were captured. Clinical studies
showed that circulation times of spherical carriers can be
extended 3-fold in humans over rats, which means the
circulation time for filomicelles in this study could reach one
month in human. It was also shown that the longer the length
of the filomicelles, the longer the circulation time they had
(Figure 24a). To test filomicelles directly as drug delivery
vehicles for cancer therapy, injections of either free drug or
drug-loaded filomicelles were given to tumor-bearing nude
mice. It was found that with increasing length of filomicelles
and increasing paclitaxel dose, cell death increases and tumor
shrinks, while little effect of free drug was shown (Figure
24b).27,114 It is worth mentioning that when the delivery of
nonspherical polymeric particles is investigated, the size of the
particles also needs to be considered because the process of
phagocytosis is a result of complex interplay between size and
shape.51
The internalization of nonspherical polymeric nanoparticles
by other nonphagocytic cells is also critical for the efficiency of
drug delivery. Nonphagocytic cells are likely to internalize
particles via endocytosis or pinocytosis pathways. Chithrani et
al. showed that the HeLa cell uptake of transferrin-coated rod-
shaped gold nanoparticles smaller than 100 nm is lower
through a citric acid ligand−receptor binding mechanism than
their spherical counterpart (Figure 25a). It was speculated that
this was due to the different curvature of spherical and rod-
shaped nanoparticles resulting in larger contact area between
the cell membrane and the longitudinal axis of the rods, and
thereby, reducing the exposure and available receptor sites.115
A further study also showed that the uptake of gold
nanoparticles of different aspect ratios by STO, HeLa, and
SNB19 cells were different, which could be explained by the
different membrane wrapping time (Figure 25b).116 Mem-
brane wrapping time was believed to be balanced by the
tension and curvature energies of the particles under the
constraint of volume, which was influenced by the size and
shape of nanoparticles.117
In another study, micro- and nanoparticles with cubic and
cylindrical shapes of various sizes were prepared with particle
replication in nonwetting templates (PRINT) method with
cross-linked poly(ethylene glycol) hydrogels. It was found that
HeLa cells internalized nanoparticles with higher aspect ratio
more rapidly than their more symmetrical counterparts with
the same volume. To delineate the role of specific endocytotic
pathways, HeLa cells were treated with biochemical inhibitors
of energy-dependent processes, clathrin-mediated, caveolae-
mediated endocytosis, and micropinocytosis. It was found the
internalization pathways for particles differing in shape and size
were biased for different particles (Figure 26). Clathrin-
mediated and caveolae-mediated endocytosis and micro-
pinocytosis played a more significant role in internalizing
smaller nanoparticles while caveolae only endocytosed nano-
particles in the range of 50−100 nm. Interestingly, the rapid
internalization of 150 nm nanoparticles with an aspect ratio of
3 might be due to the use of all internalization pathways. The
study not only demonstrated the varying efficiency of cellular
uptake for nonspherical polymeric particles with various shapes
and size, but also showed the internalization pathways of HeLa
cells.118
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To conclude, cellular interactions between nonspherical
polymeric particles with different cells, such as macrophages or
cancer cells, have been investigated and reviewed in the
literature.28,50,107,119 For drug delivery, the target is usually to
decrease the internalization by macrophages to minimize
systemic excretion so the drug-loaded nanoparticles are able to
circulate in the system for a longer duration, providing greater
chance to reach the diseased site. With diseased cells such as
cancer cells, the target is to increase accumulation and cell
killing so more internalization is preferred. Depending on the
different mechanisms of internalization by various cell types,
modifying the shape of polymeric nanoparticles is a powerful
targeting strategy for drug delivery. The studies overall
highlight the need for adequate and quality characterization
of the materials to enable comparison across studies, a point of
recent intense discussion in the literature.112
5. CONCLUSIONS AND FUTURE APPLICATIONS OF
NONSPHERICAL NANOCAPSULES
This review has systematically communicated the current
approaches to the preparation of nonspherical nanocapsules
using templates, polymerization reactions and chemical
interactions (Table 3). In conducting this review, it has been
apparent that the preparation of nanocapsules has been driven
largely from the materials chemistry standpoint and less so
from the constraints of the ultimate application. Inorganic
templates (section 3.1) were first used to prepare nonspherical
nanocapsules because they were stable throughout the
polymerization reaction and presented a ready way to maintain
the elongated shape. However, the field has largely moved on
from the use of solid templates that require the use of harsh
chemical processes to remove the underlying particle to
liberate a hollow capsule structure, toward templating using
soft materials due to the risk of damaging capsule components
or cargo. The development of nonsacrificial vesicles prepared
using surfactants or phospholipids (section 3.2) has provided a
more biologically compatible and chemically facile route to
form the polymeric shell on the surface without the need to
extract the template at the end. This aspect is extremely
important as the rigorous toxicological evaluation of new
technologies for use in drug delivery applications necessitates
the use of biologically inert components. While shape alone is
an important and useful feature described in detail, further
functionality can be coincidentally achieved through the
preparation of chemically heterogeneous particles such as
“Janus” nanocapsules (section 3.3). The ability to differentially
functionalize these materials leads to high versatility but,
conversely, complexity, which has consequences not only for
product translation but also in large scale manufacturing
contexts.
Despite the fast developments in the preparation of
nonspherical nanocapsules, three main challenges are currently
limiting advance of the field. First, only limited shapes have
been prepared and so more methods need to be developed to
enable libraries of particles changing systematically in shape
while retaining underlying chemistry and surface properties to
be prepared. In the context of drug delivery, only by creating
various shapes and understanding the complexity of their
interactions with different cells in the bloodstream and target
tissues, can the advantages of nonspherical nanocapsules be
utilized to a greater extent. Methods that enable the
preparation of a specific nonspherical morphology at higher
purity and consistency also need to be discovered to facilitate
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Chem. Mater. 2022, 34, 2503−2530
Chemistry of Materials
pubs.acs.org/cm
translation into products in the healthcare space. Second, the
reliance on polymerization reactions at high temperature also
needs to be overcome to ensure the retention and stability of
pharmaceutical actives. Research has mostly focused on
preparing different shaped nanocapsules without drug being
present, but the potential influence on the encapsulation and
activity of drugs has been neglected. Therefore, when new
methods or new shapes are being developed, considerations
need to be placed on keeping the drugs active, as well as
achieving the desired shapes. Last but not least, the
physicochemical properties of the material and its compati-
bility with the biological system need to be considered
carefully. For example, polymers with long carbon chains have
been used widely due to the ease of synthesis and greatly
available raw material. However, more biologically degradable
materials constructed using functional groups, such as amino
acid sequences and labile esters, need to be considered to avoid
accumulation and toxicity after delivery of cargo. The balance
of hydrophobicity and hydrophilicity also needs to be
considered to achieve circulation of nanocapsules to the
desired location without dramatic nonspecific protein
adsorption.
In conclusion, this review has addressed both the promises
and realities of the development of nonspherical nanocapsules.
The role that morphology plays in cellular interactions, pre-
systematic excretion, accumulation at diseased sites and
therapeutic effects is increasingly recognized but requires
more systematically varying materials of the same composition
to enable a guidance to be developed for specific applications.
■
AUTHOR INFORMATION
Corresponding Author
Ben J. Boyd − Drug Delivery, Disposition, and Dynamics,
Monash Institute of Pharmaceutical Sciences (MIPS),
Parkville, Victoria 3052, Australia; ARC Centre of Excellence
in Convergent Bio-Nano Science and Technology (CBNS),
Parkville, Victoria 3052, Australia; Department of
Pharmacy, University of Copenhagen, 2100 Copenhagen,
Denmark; orcid.org/0000-0001-5434-590X;
Email: Ben.boyd@monash.edu
Authors
Yunxin Xiao − Drug Delivery, Disposition, and Dynamics,
Monash Institute of Pharmaceutical Sciences (MIPS),
Parkville, Victoria 3052, Australia; ARC Centre of Excellence
in Convergent Bio-Nano Science and Technology (CBNS),
Parkville, Victoria 3052, Australia
Angel Tan − Drug Delivery, Disposition, and Dynamics,
Monash Institute of Pharmaceutical Sciences (MIPS),
Parkville, Victoria 3052, Australia; ARC Centre of Excellence
in Convergent Bio-Nano Science and Technology (CBNS),
Parkville, Victoria 3052, Australia; orcid.org/0000-0002-
9225-0247
Alexander W. Jackson − Functional Molecules and Polymers,
Institute of Chemical and Engineering Sciences (ICES),
Agency for Science, Technology and Research (A*STAR),
Singapore 627833; orcid.org/0000-0001-6618-7654
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.chemmater.1c03573
Notes
The authors declare no competing financial interest.
2527
■
Review
ACKNOWLEDGMENTS
The authors acknowledge the Australian Research Council
Centre of Excellence in Convergent Bio-Nano Science and
Technology for support of this work and the salary of A.T.
■
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